Tumor-selective cytotoxicity of a novel pentadiene analogue on human leukemia/ lymphoma cells

Abstract

Background: A novel series of structurally divergent 1,5-diaryl-3-oxo-1,4-pentadiene analogues 1-10 displayed marked cytotoxic potencies towards a number of human leukemia/lymphoma cells.

Objective: To identify novel selective cytotoxic compounds that induce apoptosis.

Methods: The Differential Nuclear Staining (DNS) screening protocol was utilized to measure the cytotoxicity of all experimental dienones on several cancerous cells. Additionally, the selective cytotoxicity index was calculated by comparing the dienone's cytotoxicity between leukemia/lymphoma cells vs. non-cancerous cells. Furthermore, to discern whether a selected dienone induced cell death via apoptosis or necrosis on T-lymphocyte leukemia cells, diverse approaches were utilized to detect individual biochemical facets of apoptosis.

Results: The dienones were tested for their anti-neoplastic efficiency on human leukemia/lymphoma-derived cell lines. Special emphasis was applied on dienone 1, on the basis of its sub-micromolar cytotoxicity (CC₅₀=0.43+0.02 μM) and high selective cytotoxicity index (11.1) exerted on T-leukemia cells. In general, dienone 1 showed the most potent cytotoxic properties as compared to other dienones and a related reference cytotoxin curcumin as well as the EF-24 curcumin analogue. Dienone 1 caused cell death by apoptosis in Jurkat cells as evidenced by inducing phosphatidylserine externalization, mitochondrial depolarization and caspase-3/7. These effects were mainly attributed to the induction of apoptotic pathways.

Conclusion: The novel dienone 1 was found to exhibit potent anti-leukemia activity by inducing programmed cell death/apoptosis. Consequently, dionone 1 should be developed further to examine its potential efficacy to combat malignancies in a pre-clinical animal model.

Keywords: Apoptosis; Cancer; Caspase 3; Curcumin analogue; Cytotoxins; Leukemia; Mitochondria.

Fig. (1)

Structures of compound 1 to 10, EF-24 and Curcumin.

Fig. (2)

Analysis of the cytotoxic properties of various 1,5-diaryl-3-oxo-1,4-pentadienes 1–10 against hematological malignancies. The cytotoxic potential of 10 curcumin analogues was studied in 13 cell lines by DNS. The compounds were tested at a final concentration of 1 μM and incubated for 20 h. For ease of presentation, a heat map was used to depict the results that shows a gradient from green (no toxicity) to red (100% toxicity) with black representing ~50% cytotoxicity. The most affected cell lines (red squares) were Ramos, Nalm-6, Jurkat, and CEM. The two non-transformed cell lines tested, MCF-10A and Hs-27, were not significantly affected by the test compound after 20 h of exposure. Note that 1 at the top of the heat map was selected for further characterization as it exhibited cytotoxic activity against the majority of leukemia/lymphoma cell lines but not the non-transformed cell lines. The MeV heat-map generating software was used to construct the heat maps.

Fig. (3)

Compound 1 induced significant phosphatidylserine (PS) externalization on Jurkat and Nalm-6 leukemia cells after 24 h of treatment. PS exposure on the cell surface was monitored after treatment of two distinct lymphoma lines, Jurkat (a) and Nalm-6 (b). Apoptosis and necrosis were examined after incubation of the cell lines and co-staining with annexin V-FITC and PI and analyzed via flow cytometry assay. Each bar represents the average of three replicas with standard deviations. The total percentage of apoptotic cells is displayed as the sum of annexin V-FITC positive cells at early and late stages of apoptosis (white bars). PI-positive cells, but negative for annexin V-FITC, are considered undergoing necrosis (black bars). The following controls were included in these assays: treatment with the reference drugs curcumin and the EF-24 analogue (both at 1 μM); untreated cells as a negative control; DMSO as the solvent control; and H₂O₂-treated cells as a positive control for cytotoxicity. For both cell lines, a comparison of 1-treated cells with untreated (*) and DMSO (‡) controls, consistently resulted in P-values lower than 0.001. P-values comparing the percentage of apoptosis induced by 1 as compared with curcumin are annotated in the graphs. Data acquisition, processing and analysis were achieved by utilizing CXP software (Beckman Coulter).

Fig. (4)

Compound 1 induced mitochondrial depolarization and capase-3/7 activation in Jurkat cells. All assays were performed by flow cytometry and approximately 10,000 events (cells) were acquired for each sample. DMSO (1% v/v)-treated and untreated (Unt) cells were included in all three assays as controls. (a) JC-1 polychromatic fluorophore was used as a mitochondrial transmembrane potential indicator, after 7 h of exposure. Cells treated with the proton ionophore, 50 μM carbonyl cyanide m-chlorophenyl hydrazone (CCCP), were used as the positive control for mitochondria depolarization. (b) Intracellular active caspase-3/7 was identified after 20 h of incubation via NucView 488 fluorogenic substrate; the percentage of active caspase 3/7-positive cells, exhibiting green fluorescence signal, is indicated on the y-axis. Camptothecin (CPT; 2 μg/ml) was used as positive control. P values (***) were consistently below 0.001 when 1-treated cells were compared to DMSO-treated cells as evaluated by using Student’s t-test in all three assays. Data acquisition, processing and analysis were performed using CXP software (Beckman Coulter). Each bar represents the average of triplicates and the error bars represent the standard deviation of the mean.